CN113321707A - 一种人工合成抗菌肽及其应用 - Google Patents
一种人工合成抗菌肽及其应用 Download PDFInfo
- Publication number
- CN113321707A CN113321707A CN202110364359.7A CN202110364359A CN113321707A CN 113321707 A CN113321707 A CN 113321707A CN 202110364359 A CN202110364359 A CN 202110364359A CN 113321707 A CN113321707 A CN 113321707A
- Authority
- CN
- China
- Prior art keywords
- antibacterial peptide
- peptide
- antibacterial
- lysine
- lys
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- PEFJUUYFEGBXFA-BZSNNMDCSA-N Phe-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CC1=CC=CC=C1 PEFJUUYFEGBXFA-BZSNNMDCSA-N 0.000 claims description 6
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/001—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof by chemical synthesis
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3526—Organic compounds containing nitrogen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
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Abstract
本发明提供了一种人工设计合成抗菌肽的序列及其制备方法,本发明还提供了不同构型的该抗菌肽在抗微生物中的应用;本发明抗菌肽具有显著的抑制细菌、真菌生长的作用;本发明抗菌肽具有结构简单、人工合成方便、抗菌谱系广的有益特点;其全D‑型及碱性氨基酸由D‑型氨基酸组成分子具备耐受多种蛋白酶的降解,抗菌活性保留时间长,无药物残留的特性;可应用于人或动物感染性疾病的预防和治疗、人工生物材料、食品防腐、果蔬保鲜、饲料添加剂中。
Description
技术领域
本发明提供了一种人工设计合成的抗菌肽,本发明还涉及该抗菌肽的制备及其应用,属生物医学领域。
背景技术
抗菌肽是存在于生物体内能抵抗外界微生物侵害的一类小分子多肽,它是先天免疫系统的重要组成部分。抗菌肽通常由l5~45个氨基酸残基组成,大部分带正电荷;抗菌肽广泛地分布在两栖类、昆虫、植物和哺乳动物中。与目前广泛使用的抗生素相比,抗菌肽具有很多优点:如在最小作用浓度时,快速而广谱的杀灭微生物活性;不易产生耐药性;热稳定、水溶性好;对局部感染和全身感染都有效等;长期以来,抗生素在抗菌临床应用上见效快、疗效显著,成为治疗细菌性疾病的首选药物;然而,随着抗生素的大规模使用,人们发现,常用抗生素的治疗效果逐渐减弱,与之对应的是,病原菌耐药性不断增强,导致抗生素的单位用药量不断加大,抗生素的残留问题也愈发严重,威胁着人类健康,抗菌肽作为一种抗生素的替代物备受关注。
抗菌肽具有广谱抗微生物活性,对革兰氏阳性和革兰氏阴性细菌、真菌和被膜病毒都有抑杀作用,特别对一些耐药菌,如人病原菌伤寒杆菌、金黄色葡萄球菌和水产养殖病原菌嗜水气单胞菌、维氏气单胞菌、溶藻弧菌、霍乱弧菌等同样敏感。其作用机制主要是带正电的抗菌肽与带负电的微生物细胞膜发生作用,按作用特点可分为膜结构破坏型和非膜结构破坏型。在当前不少病原菌对原有抗生素逐步产生耐药性的情况下,加强对动物体内自然产生的抗菌肽功能的了解,并在此基础上设计新型的抗菌肽以替代抗生素的研究有着重要的理论和实际意义。
目前发现的天然存在的抗菌肽分子大多数是由L-型氨基酸残基组成,因分子中含有较多的碱性氨基酸残基而易受到各种蛋白酶的降解,影响了它们的抗菌活性。本发明设计合成了一个新的抗菌肽,其D-型氨基酸残基组成抗菌肽在保留了抗菌活性的同时,不易受到蛋白酶的降解,有效延长了作用时间。
发明内容
本发明的目的是提供一种抗菌肽及其不同构型分子在抗微生物中的应用。非天然全L-型或D-型氨基酸组成的抗菌肽具有显著的抑制细菌以及真菌生长的作用,尤其是对不同耐药细菌有很好的抑制作用,且具有结构简单、人工合成方便、抗菌谱系广的有益特点。其显著特点在于D-型氨基酸组成的抗菌肽能耐受各种蛋白酶的降解,抗菌活性保留时间长,无药物残留。可以应用于人或动物感染性疾病的预防和治疗、人工生物材料、食品防腐及果蔬保鲜、饲料添加剂中。
为了实现本发明的目的,本发明提供了如下技术方案:
人工设计合成抗菌肽,所述抗菌肽(Peptide 3)具有以下序列:
SEQ ID NO:1:Lys Lys Phe Lys Lys Phe Phe Lys Lys Leu Leu Lys Ser ValLys Lys Lys Phe Lys Lys Phe Phe Lys Lys Leu Lys Val Ile Gly Val Thr Phe ProPhe;
组成该抗菌肽的氨基酸残基可为D-型或L-型氨基酸残基,尤其是碱性氨基酸赖氨酸残基。
人工设计合成抗菌肽的制备方法:采用全自动多肽合成仪合成,按照抗菌肽全序列(SEQ ID NO:1),可合成全部由L-型、D-型氨基酸残基组成,或碱性氨基酸赖氨酸残基为D-型氨基酸残基而其余氨基酸残基为L-型氨基酸残基组成的合成分子。通过HPLC反相C18柱层析脱盐、纯化。然后用HPLC方法鉴定其纯度,分子量测定采用快原子轰击质谱法(Fastatom bombardment mass spectrometry,FAB-MS)。
人工设计合成抗菌肽作为制备抗微生物物质的应用:
人工设计合成抗菌肽具有显著的抑制细菌以及真菌生长的作用,尤其是对临床及水产耐药细菌有很好的抑制作用。与其它来源抗菌多肽相比,该抗菌肽具有结构简单、人工合成方便、抗菌谱系广的有益特点,尤其是含D-型氨基酸残基分子具有抵抗蛋白酶水解作用的显著特点,可更长时间保持抗微生物活性,无药物残留。
具体实施方式
实施例 1 人工设计合成抗菌肽的制备:
1、分别用全自动多肽合成仪利用不同构型氨基酸残基为原料合成全D-型抗菌肽、全L-型抗菌肽以及碱性氨基酸赖氨酸残基为D-型氨基酸残基而其余氨基酸残基为L-型氨基酸残基组成的合成分子的全序列。通过HPLC反相C18柱层析脱盐、纯化。
2、分子量测定采用快原子轰击质谱法(Fast atom bombardment massspectrometry, FAB-MS),以甘油:间硝基苄醇:二甲亚砜 (1:1:1, V:V:V,体积比)为底物,Cs+作为轰击粒子,电流为1电微安,发射电压为25 Kv。
3、纯化的抗菌肽用高效液相色谱HPLC方法鉴定其纯度,分子量测定采用快原子轰击质谱法。
所合成抗菌肽的序列如下所示:
Peptide 34:SEQ ID NO:1:赖氨酸-赖氨酸-苯丙氨酸-赖氨酸-赖氨酸-苯丙氨酸-苯丙氨酸-赖氨酸-赖氨酸-亮氨酸-亮氨酸-赖氨酸-丝氨酸-缬氨酸-赖氨酸-赖氨酸-赖氨酸-苯丙氨酸-赖氨酸-赖氨酸-苯丙氨酸-苯丙氨酸-赖氨酸-赖氨酸-亮氨酸-赖氨酸-缬氨酸-异亮氨酸-甘氨酸-缬氨酸-苏氨酸-苯丙氨酸-脯氨酸-苯丙氨酸 (Lys Lys Phe Lys Lys Phe Phe Lys Lys Leu Leu Lys Ser Val LysLys Lys Phe Lys Lys Phe Phe Lys Lys Leu Lys Val Ile Gly Val Thr Phe Pro Phe);
实施例2 不同构型抗菌肽抑制细菌生长的作用:
抗菌活性检测,按常规倍比稀释法进行。96孔板每空加菌液90微升(终浓度2×104CFU/ml),每孔设3个复孔,以pH7.0 LB培养基(每升含10g胰蛋白胨,Oxoid公司生产;5g酵母提取物,Oxoid公司生产;10g氯化钠,国产分析纯试剂)为阴性对照。加入不同浓度的用无菌超纯水配制的抗菌肽10微升,使终浓度成倍比下降。37 ℃孵育14-16h,测定各孔600nm的吸光度,吸光度无变化的最低浓度为最小抑菌浓度(MIC)。为测试高盐条件下抗菌肽对海洋细菌的作用,高盐pH7.0 LB培养基(每升含10g胰蛋白胨,Oxoid公司生产;5g酵母提取物,Oxoid公司生产;60g氯化钠,国产分析纯试剂)用于欧文斯氏弧菌、轮虫弧菌和需钠弧菌的抑菌实验。试验重复3次,取平均值,结果如表1。
实验所用细菌以及真菌菌株说明:
金黄色葡萄球菌(Staphyloc occus aureus)ATCC 43300,是耐甲氧西林的金黄色葡萄球菌ATCC标准株,对青霉素类,b-内酰胺类耐药,对万古霉素敏感。
大肠杆菌(Escherichia coli) ATCC 25922为不耐药标准株。
大肠杆菌(Escherichia coli)临床耐药株1和2,均为产超广谱β-内酰胺酶的临床分离株,对青霉素,头孢1代、2代、3代以及4代耐药,对泰能,氨基糖甙类敏感。
绿脓杆菌(Pseudomonas aeruginosa)临床耐药株,为多药耐药临床分离株,对目前临床使用各类抗生素均耐药,包括泰能,MEM。
真菌类的白色念珠菌Candida albicans ATCC 10231为标准株。
其余所用菌株采用细菌16S rDNA菌种鉴定方法:提取培养单克隆菌株DNA,特定引物PCR扩增,PCR产物纯化测序,进化树分析精确鉴定到种。
由表1可见,不同构型本发明抗菌肽具有显著的抑制细菌以及真菌生长的作用,可作为抗微生物物质用于制备抗微生物感染制剂。特别是在高盐培养条件下仍然显示极强的针对欧文斯氏弧菌、轮虫弧菌和需钠弧菌的抗菌活性。
表1 不同构型抗菌肽Peptide 34的抗微生物活性
实施例3 人工设计合成抗菌肽抗蛋白酶水解作用:
实验方法:将采购自Sigma公司的胰蛋白酶、糜蛋白酶、胃蛋白酶以及弹性蛋白酶按酶:抗菌肽=1:10(摩尔比)的比例混合后,37 ℃孵育24 h后进行大肠杆菌的抗菌活性测定,抗菌肽的终浓度均为400 ug/ml。抗菌活性检测采用琼脂糖打孔法进行,培养基配方为:1%低熔点琼脂糖(Sigma A6013),0.3 mg/ml胰蛋白冻(Oxoid产品)溶于10 mM pH 7.4Na2HPO4- NaH2PO4缓冲液中。制备的培养基20 ml于42℃时分别加入过夜培养对数生长期大肠杆菌ATCC 25922(约105-106CFU),摇匀后使其在直径76mm培养皿中均匀摊布。待凝固后,培养基上打3 mm的圆孔,每孔分别加10 ul样品后37℃继续孵育16 h后,测量无菌生长的透明环直径进行抗菌活性的测定。抗菌活性的计算:抗菌活性单位U=(抗菌环的直径mm-3)×10。37℃孵育24 h的相同浓度抗菌肽和蛋白酶分别作为阳性对照和阴性对照,37 ℃孵育24 h的酶+抗菌肽孵育液为实验组,结果见表2。
表2 抗菌肽与蛋白酶37℃保温24 h后的抗菌活性单位
由表2可见,全D-型抗菌肽与胰蛋白酶、糜蛋白酶、胃蛋白酶以及弹性蛋白酶按酶:抗菌肽=1:10(摩尔比)的比例混合后,37 ℃孵育24 h后抗菌活性保持不变,揭示了多种蛋白酶不能水解全D-型抗菌肽。而碱性氨基酸Lys全为D-型和全L-型抗菌肽的抑菌活性受蛋白酶的影响较大。
实施例4 人工设计合成抗菌肽对绿脓杆菌角膜感染的保护作用
实验方法:新西兰大白兔平均2.5 Kg/只,每组3只。绿脓杆菌(ATCC 27853)在LB培养基中过夜培养,并重新接种培养至对数生长期,离心收集细菌用无菌生理盐水调整细菌数目为105 CFU/ml。兔子用2%的戊巴比妥钠经耳缘静脉全身麻醉,盐酸普鲁卡因于眼眶外局部麻醉后,用手术镊触及角膜无反应,将异丙醇20ul滴于直径为0.5 cm的角膜环钻内30s,造成角膜上皮损伤,吸去异丙醇,用生理盐水冲洗角膜。将调整好浓度的绿脓杆菌30 ul滴于损伤的角膜中心,按上述方法处理后,实验动物100%发生感染。接种细菌于角膜12 h后,开始滴眼治疗。左眼为不同构型抗菌肽(4mg/ml),右眼为生理盐水对照,每次10ul,实验前3天每天进行6次治疗,此后每天早晚各进行1次治疗。最后1次滴眼后4 h处死兔子,用角膜环钻取大小相同的角膜,称重。用生理盐水冲洗3次后,用微量匀浆器5000 r/min,匀浆3次,每次30 s。将匀浆液按10倍稀释法稀释后,分别进行LB平板培养计数。每个抗菌肽用3只兔子进行平行对照实验,结果见表3。
表3 抗菌肽及其衍生物对绿脓杆菌角膜感染的保护作用
由表3可见,不同构型抗菌肽及其衍生物对绿脓杆菌引起的角膜感染具有显著的保护作用,可应用于抗眼部微生物感染制剂的制备。
实施例5 不同构型抗菌肽与抗生素的协同作用:
实验方法:临床分离鉴定的鲍氏不动杆菌菌株由昆明医科大学第一附属医院检验科提供,注射用头孢哌酮钠为哈药集团制药总厂生产。按照实施例2所述抗菌活性检测方法得到的头孢哌酮钠对该菌株的MIC值为32 ug/ml,不同构型抗菌肽对该菌株的MIC值见表4。根据以上结果,固定不同构型抗菌肽的剂量为MIC值的一半(1/2 MIC),加入不同浓度的头孢哌酮钠,测定完全抑制细菌生长所需的抗生素浓度。实验重复3次,每次3孔,结果见表4。
表4 不同构型抗菌肽与抗生素抑制临床鲍氏不动杆菌生长的协同作用
由表4可见,不同构型抗菌肽与临床抗生素对微生物的抑制作用有显著的协同性,有助于抗菌肽与现有抗生素联合应用用于人或动物感染性疾病的预防和治疗。
实施例6 不同构型抗菌肽对人红细胞的溶血活性:
实验方法:健康人红细胞购自昆明市中心血站,人红细胞与0.38%柠檬酸钠按1:9(v/v)的比例混合后,2000 r/min离心5min,弃上清。然后用生理盐水充分洗涤人红细胞至不再呈红色为止。将上述洗涤好的人红细胞加生理盐水稀释成约108浓度的悬浮液。上述稀释好的悬浮液与溶解于生理盐水的不同浓度的样品于37°C保温1小时,2000 r/min离心5min后于540nm测定上清液的吸收值,溶血活性根据各样品与加入1%的Triton X-100得到的最大溶血值相比而确定,结果见表5。
结果表明:不同构型抗菌肽在浓度为50-200 ug/ml时,基本不具有对人红细胞的溶血活性。
实施例7 不同构型抗菌肽在人工生物材料中的应用
本实施例实验材料来源:医用涤纶补片为北京佰仁医疗科技有限公司产品;医用几丁糖凝胶为石家庄亿生堂医用品有限公司产品;注射用头孢哌酮钠为哈药集团制药总厂生产;一次性使用气管插管为博罗其乐康医疗用品有限公司产品;兔子购自昆明医科大学动物房。
7.1 全D-型抗菌肽在医用涤纶补片及医用几丁糖凝胶中的应用
兔涤纶补片细菌感染模型的构建及实验结果:
实验方法: 兔6只,每只兔背部皮肤手术切开四个1cm2大小袋状空隙。将医用涤纶补片剪成1×1cm小块,分为四组,分别处理。空白组:不做任何处理;几丁糖组:均匀涂布几丁糖,70℃固化1h;抗生素组:均匀涂布几丁糖+抗生素(头孢哌酮),10 mg/ml,70 ℃固化1h;抗菌肽组:均匀涂布几丁糖+抗菌肽,200 ug/ml,70 ℃固化1h。将处理过的医用涤纶补片分别植入袋状空隙中,向内注入1ml (含2×107cfu大肠杆菌)菌液,缝合切口。7天后处死兔子取出涤纶补片进行悬浮和紧密附着于涤纶补片的细菌计数。全D-型抗菌肽(SEQ IDNO:1)的实验结果如表6、表7所示:
*说明:涤纶补片洗2遍后,经铺板培养后菌落数为0,说明洗涤2遍后涤纶补片无游离的细菌残留,震荡1分钟后产生的细菌为牢固附着于涤纶补片的产生了生物膜(Biofilm)样结构的细菌。
实验结果表明:医用涤纶补片经抗菌肽处理后,能显著降低大肠杆菌的附着,降低生物膜(Biofilm)的形成,有助于预防临床生物材料相关感染的治疗。200 ug/ml抗菌肽处理医用涤纶补片的效果相当或优于10 mg/ml头孢哌酮。但本发明抗菌肽对大多数临床耐药株有效,揭示了OH-CATH30-D可应用于临床医用涤纶补片类人工生物材料。同时,抗菌肽组(几丁糖+抗菌肽)与几丁糖组(仅加几丁糖)比较,抗菌肽组对微生物的抵抗作用显著优于几丁糖组。因此,本发明抗菌肽也可应用于临床医用凝胶类人工生物材料。
在兔涤纶补片细菌感染模型中,Peptide 34 Lys全D-型和Peptide 34全L-型抗菌肽也显示了类似的实验结果,表明本发明抗菌肽可应用于人工生物材料如临床医用涤纶补片类以及临床医用凝胶类中防治微生物感染。
7.2全D-型抗菌肽在医用导尿管中的应用
兔导尿管细菌感染模型的构建及实验结果
实验方法:实验共分四组,空白组、几丁糖组、抗生素组、抗菌肽组,每组兔子6只。用双腔导尿管(杭州富善医疗器械有限公司产品)留置导尿,1 N的盐酸酸化处理膀胱,24h后更换为已处理的导尿管,并注入OD600nm =1时稀释107的大肠杆菌1ml,夹闭导尿管1h。导尿管的处理方法:空白组:不做任何处理;几丁糖组:均匀涂布几丁糖,70℃固化1h;抗生素组:均匀涂布几丁糖+抗生素(10 mg/ml头孢哌酮),70℃固化1h;抗菌肽组:均匀涂布几丁糖+抗菌肽(200 ug/ml),70 ℃固化1h。分别于0,1,5,10天留尿,第10天处死实验动物后将导尿管取出,分为头段和中段,每段长约1cm,洗涤2次,震荡1min,取液体50 ul铺板培养测定菌落数。全D-型抗菌肽处理导尿管的实验结果见表8-表12所示。
实验结果表明:医用导尿管经抗菌肽处理后,能显著降低大肠杆菌的附着,降低生物膜(Biofilm)的形成,有助于预防临床生物材料相关感染的治疗。200 ug/ml抗菌肽处理医用导尿管的效果优于10 mg/ml头孢哌酮。但抗菌肽对大多数临床耐药株有效,揭示了本发明抗菌肽可应用于临床医用导尿管类人工生物材料。同时,抗菌肽组(几丁糖+抗菌肽)与几丁糖组(仅加几丁糖)比较,抗菌肽组对微生物的抵抗作用显著优于几丁糖组。因此,本发明抗菌肽也可应用于临床医用凝胶类人工生物材料。
实施例8 人工设计合成抗菌肽在食品防腐及果蔬保鲜中的应用:
8.1 不同构型抗菌肽在食品防腐中的应用
实验方法:昆明德和罐头厂生产红烧牛肉罐头购自超市,所有实验操作均在实验室非无菌条件下进行。开罐后将罐头内容物全部取出,机器匀浆后于10000 r/min离心10min,取上清液备用。实验组为900 ul上清液+50 ul抗菌肽(终浓度200 ug/ml)+50 ul大肠杆菌临床耐药株2(终浓度105 CFU/ml),对照组为900 ul上清液+50 ul灭菌超纯水+50ul大肠杆菌临床耐药株2(终浓度105 CFU/ml),然后置于培养箱25℃进行培养,观察并记录48h培养液发生的变化。实验结果见表13。
结果表明:罐头食品内含物经不同构型抗菌肽抗菌肽处理后,能显著增强罐头食品内含物对以大肠杆菌为代表的环境微生物的抵抗能力,不同构型抗菌肽无药物残留问题,可用于食品防腐。
8.2 不同构型抗菌肽在果蔬保鲜中的应用
实验方法:新鲜杨梅及荔枝购自超市,所有实验操作均在实验室非无菌条件下进行。杨梅经超纯水充分洗涤后,随机分为实验组与对照组,10个/组。实验组杨梅用终浓度为200 ug/ml的抗菌肽溶液浸泡30min,对照组杨梅用超纯水浸泡30 min,然后置于培养箱25℃进行培养,观察并记录72 h时杨梅发生的变化。荔枝小心去皮后,随机分为实验组与对照组,10个/组。实验组荔枝用终浓度为200 ug/ml的抗菌肽溶液浸泡30 min,对照组杨荔枝用超纯水浸泡30 min,然后置于培养箱25 ℃进行培养,观察并记录72 h时荔枝发生的变化。以上实验结果见表14。
结果表明:新鲜水果经不同构型抗菌肽处理后,能显著增强新鲜水果对环境微生物的抵抗能力,可用于果蔬保鲜。
实施例9 不同构型抗菌肽在饲料添加剂中的应用:
实验方法:颗粒饲料购自昆明医科大学动物科,所有实验操作均在实验室非无菌条件下进行。颗粒饲料经研磨后,按1:1(重量/体积)用灭菌生理盐水溶解,10000 r/min离心10 min,取上清液备用。实验组为900 ul上清液+100 ul抗菌肽(终浓度200 ug/ml),对照组为900 ul上清液+100 ul灭菌超纯水。然后置于培养箱25 ℃进行培养,72 h后用显微镜观察培养液,实验结果见表15。
结果表明:动物饲料经不同构型抗菌肽处理后,能显著增强动物饲料对环境微生物的抵抗能力,不同构型抗菌肽具备抗细菌及抗真菌的活性,无药物残留问题,可作为饲料添加剂。
Claims (7)
1.一种人工设计合成抗菌肽,其特征在于所述抗菌肽为选自序列表 SEQ ID NO:1所示的氨基酸序列(Peptide 34):
SEQ ID NO:1:赖氨酸-赖氨酸-苯丙氨酸-赖氨酸-赖氨酸-苯丙氨酸-苯丙氨酸-赖氨酸-赖氨酸-亮氨酸-亮氨酸-赖氨酸-丝氨酸-缬氨酸-赖氨酸-赖氨酸-赖氨酸-苯丙氨酸-赖氨酸-赖氨酸-苯丙氨酸-苯丙氨酸-赖氨酸-赖氨酸-亮氨酸-赖氨酸-缬氨酸-异亮氨酸-甘氨酸-缬氨酸-苏氨酸-苯丙氨酸-脯氨酸-苯丙氨酸 (Lys Lys Phe Lys Lys Phe Phe Lys Lys Leu Leu Lys Ser Val Lys Lys Lys PheLys Lys Phe Phe Lys Lys Leu Lys Val Ile Gly Val Thr Phe Pro Phe)。
2.权利要求1所述的抗菌肽,其特征在于:组成该抗菌肽的氨基酸残基可为D-型或L-型氨基酸残基,尤其是碱性氨基酸赖氨酸残。
3.权利要求1-2所述抗菌肽及其衍生物的制备方法,其特征在于:采用固相化学法合成。
4.权利要求1-2所述抗菌肽的应用,其特征在于:所述抗菌肽可单独用于制备人或动物抗微生物感染药物或所述抗菌肽与现有药物联合应用,用于人或动物感染性疾病的预防和治疗。
5.权利要求1-2所述抗菌肽的应用,其特征在于:所述抗菌肽在人工生物材料中的应用。所述人工生物材料可以是但不局限于医用高分子生物材料、医用金属材料、医用生物复合物、医用生物衍生材料和医用凝胶。
6.权利要求1-2所述抗菌肽的应用,其特征在于:所述抗菌肽在食品防腐及果蔬保鲜中的应用。
7.权利要求1-2所述抗菌肽的应用,其特征在于:所述抗菌肽在饲料添加剂中的应用。
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